This invention relates to a process for making reagents used in the conduction of agglutination reactions. More particularly this invention relates to a process for forming such reagents useful in a process for accurately measuring the concentration of agents that promote or inhibit agglutination reactions, and wherein the extent of the agglutination reaction is determined by quasi-elastic light scattering spectroscopy or other optical methods.
Agglutination reactions are widely used in biology and medicine to detect small quantities of antibody or antigen molecules. Agglutination reactions usually involve the in vitro aggregation of microscopic carrier particles which bear on their surface antigenic molecules. Aggregation occurs when antibody molecules specifically corresponding to the antigen are introduced into the solution of the carrier particles. The converse procedure of agglutinating antibody-coated particles with the appropriate polyhaptenic antigen molecules is also used. Some of the carrier particles which have been used are red blood cells, bacteria and polystyrene spheres. At low concentrations of the agglutination-inducing antibody or antigen (henceforth termed the agglutinator), small aggregates consisting of only a few carrier particles are formed. At higher concentrations of agglutinator the aggregates grow so large as to form visible clumps.
Conventionally, the appearance of this visible agglutinate has been taken as the criterion for the presence of the agglutinator. Clearly, this detection criterion suffers from several defects. First, the formation of the grossly visible agglutinate requires a much larger concentration of agglutinator than needed to form small microscopic aggregates. Moreover, whereas the reversible formation of small aggregates is a specific and reproducible process, the appearance of macroscopic agglutinates is subject to many poorly controlled influences, such as the presence of foreign surfaces. In addition, the appearance of a grossly visible agglutinate is so qualitative a criterion that it is difficult experimentally to determine quantitatively the associated agglutinator concentration. Conventionally, the agglutinator concentration is determined by preparing a serial dilution of the agglutinator-containing solution. Then an aliquot of each dilution is mixed with a fixed amount of carrier particles (henceforth all the reagents, including carrier particles, used in fixed amount will be collectively termed the agglutinant) and the highest degree of dilution which still permits the formation of a visible agglutinate, is noted. This serves to indicate the concentration of agglutinator in the original solution. The agglutinator concentration can at best be determined to within a factor of two by this method.
Thus, while the agglutination reaction, as conventionally performed serves as a specific and versatile means of detecting antigen or antibody molecules, it is severely limited in its application in that: (1) the process is not capable of providing an accurate quantitative measurement of either antigen or antibody concentrations and (2) the process may only be used for determining antibody or antigen concentrations which are sufficiently high so as to induce (or inhibit) macroscopically visible agglutination.
In copending patent application Ser. No. 662,497 filed Mar. 1, 1976 entitled "Immunoassay by Light Scattering Spectroscopy", there is described a process for measuring the concentration of substances capable of promoting or inhibiting agglutination reactions which is substantially more accurate and sensitive than previously known processes used for determining the extent of agglutination reactions.
In the process, the degree of agglutination is determined by measuring the mean diffusion constant D of the agglutinated reaction product by means of quasi-elastic light scattering spectroscopy. The measured diffusion constant then is compared with a standard quantitative relationship between the mean diffusion constant of the agglutinated reaction product and the concentration of antigen or antibody being tested. In addition to determining the concentration of antigen or antibody molecules, the process can be used to determine the concentration of any substance capable of specifically promoting or inhibiting an agglutination reaction even when the formation of antigen-antibody bonds is not involved in the agglutination process.
Quasi-elastic light scattering spectroscopy is a laser technique used to study the Brownian motion of particles in solution. The Brownian motion of a particle in solution is characterized by its diffusion constant D, which is a monotonically decreasing function of particle size. This Brownian motion broadens the spectral linewidth of the laser light scattered by the particle in direct proportion to D. The spectrum (or its Fourier transform, the correlation function) of the light scattered from a polydisperse solution of particles is determined by the distribution of diffusion constants of the particles. By analysis of the spectrum (or correlation function) D is obtained which is the average of the diffusion constants of the particles weighted by the intensity of the light scattered by each particle. The mean diffusion constant D is inversely proportional to the mean hydrodynamic radius of the particles. The appearance of even a few aggregates in a previously monodisperse solution of particles causes a marked drop in D because the aggregates scatter light more strongly than do the single particles. The scattered light is measured at a constant angle to the path of incident light. Quasi-elastic light scattering is a particularly suitable means of following agglutination in that it measures only the relative distribution of particle sizes, and is independent of absolute particle concentration. Hence, quasi-elastic scattering measurements are insensitive to the anomalies of precipitation and absorption of particles onto foreign surfaces.
In the process, an agglutination reaction is performed in any of a variety of modes of operation. The agglutination reaction may be used in several different modes to detect antigen or antibody including procedures which utilize carrier particles as follows:
(1) With antigen-coated carrier particles as agglutinant and the complementary antibody as agglutinator.
(2) With antibody-coated carrier particles as agglutinant and the complementary antigen as agglutinator.
(3) The agglutination inhibition mode with antigen-coated spheres wherein a fixed quantity of antibody is mixed with a dilution of the test sample containing the complementary antigen, inactivating a portion of the antibody. This mixture then is combined with the antigen-coated carrier particles. The degree to which the antigen in the test sample inhibits the aggregation of the carrier particles, that would otherwise have occurred, indicates the concentration of antigen present.
(4) The agglutination inhibition mode with antibody-coated spheres wherein a fixed quantity of antigen is mixed with a dilution of the test sample containing the complementary antibody, inactivating a portion of the antigen. This mixture then is combined with the antibody-coated carrier particles. The degree to which the antibody present in the sample inhibits the aggregation of carrier particles, which would otherwise have occurred, indicates the concentration of antibody present.
In modes 1 and 4 the agglutination reaction serves as an antibody assay. In modes 2 and 3 it serves as an antigen assay. Mode 3 is of particular practical importance as an antigen assay since it is generally easier to obtain a sufficient quantity of purified antigen to coat the carrier particles than to obtain a similar quantity of complementary antibody. Moreover, in mode 3 the agglutination reaction serves to detect antigen molecules of any size with one or more haptenic sites. On the other hand, in mode 2 the agglutination reaction serves to detect only polyhaptenic antigens, which are of sufficient size (on the order of 100 A in diameter) to effect crosslinking of the carrier particles.
A standard quantitative relationship first is established between the mean diffusion constant D of the agglutinated reaction product as a function of the concentration of the antigen or antibody being tested for fixed concentrations of the agglutinant composition. Antigen or antibody-coated particles can be prepared by depositing the antigen or antibody of the surface of latex microspheres, red blood cells, bacteria or the like by means well known in the art. In addition, some cells or bacteria naturally bear certain antigens or antibodies on their surface. Serial dilutions of known concentration of the antigen or antibody one wishes to test then are prepared and an agglutination reaction is performed using these serial dilutions of known concentration of antigen or antibody with the fixed concentrations of the agglutinant composition. The concentration of agglutinator present must be sufficiently low so that precipitation of the agglutinated particles does not occur, so that the agglutinated particles remain suspended in solution. The agglutination reaction involves the cross-linking of the coated particles to produce larger particles in proportion to the concentration of active agglutinator present. This cross-linking has the effect of reducing the mean diffusion constant measured by quasi-elastic light scattering spectroscopy in proportion to the concentration of active agglutinator present. For each serial dilution of the known concentration of antigen or antibody tested, the value of D is determined for the corresponding agglutinated reaction product by means of quasi-elastic light scattering spectroscopy as described above.
The quantitative relationship so-determined then can be employed as a standard to be applied when performing the agglutination reaction on samples containing unknown amounts of the antigen or antibody being tested. A serial dilution of each sample is prepared. The agglutination reaction is performed using one or several of these dilutions of the sample and the mean diffusion constant D is determined. The agglutinant composition employed to establish the standard quantitative relationship must be the same agglutinant composition employed to form the agglutinated reaction product with the antigen or antibody being tested so that an accurate comparison can be made between the standard and the unknown. The measurements of D of the agglutinated reaction product obtained with the antigen or antibody being tested are compared with the standard quantitative relationship between D and the antigen or antibody concentration, and thus the original concentration of the antigen or antibody in the sample is determined. At least two sample dilutions should be analyzed by light scattering spectroscopy in order to extrapolate the results to the standard quantitative relationship. However, only one sample dilution is needed if it is known that, at the concentration tested, it is within an ascending or descending portion of the relationship between concentration and mean diffusion constant.
It is also possible to use the process to measure the concentration of antigen or antibody without the use of carrier particles. Thus, in addition to the modes of operation set forth above, the concentration of antigen is determined by mixing a fixed amount of complementary antibody to the serial dilutions of the sample which contains the antigen (mode 5). Quasi-elastic light scattering then is used to measure D of the reaction product and this is compared with standard quantitative relationship. The larger the antigen-antibody aggregates the smaller is D. In mode 6 of the method, antibody concentration is determined by mixing a fixed amount of complementary antigen with the serial dilutions of the sample containing the antibody. Once again D of the reaction product is measured and is compared with a standard quantitative relationship. In practice, the process is more sensitive in modes 1, 2, 3, 4 where carrier particles are utilized as compared to modes 5 and 6. Also, there is less interference in the measurement of D due to light scattered by other elements present in the sample in modes 1, 2, 3, 4 as compared to modes 5 and 6 since the intensity of light scattered by the large carrier particles is generally much greater than that scattered by any other element. However, modes 5 and 6 are useful when it is not possible to bind the antigen or antibody to a suitable carrier particle.
The process provides substantial advantages over the processes of the prior art. Since the process does not require that the agglutination reaction be conducted at such a high concentration of agglutinator and agglutinant that macroscopic precipitation of the agglutination reaction product occurs. Thus, the method can be used to measure much lower antigen or antibody concentrations associated with the microscopic reversible stages of the agglutination reaction. This stage may involve the dimerization of the carrier particles whereas the macroscopically visible agglutinate may contain millions of carrier particles. Moreover, in the process the degree of agglutination is quantatively measured at the microscopic, reversible and reproducible stage of the agglutination reaction. Thus this process serves to transform the agglutination reaction from a rough qualitative measure of antigen or antibody concentration to an accurate, reproducible means of quantitating antibody or antigen concentrates.
It would be highly desirable to provide a means for optimizing the sensitivity, specificity, accuracy and reproducibility of the immunoassay by light scattering spectroscopy procedure, by optimizing the composition of the reagents used in the procedure. These reagents may comprise antigen, antibody, antigen-coated particles, antibody-coated particles, buffered solvents, various organic and inorganic salts, viruses, red blood cells, bacteria and the like.